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Open AcceReviewCyanobacterial lipopolysaccharides and human health – a reviewIan Stewart*1,2,3, Philip J Schluter4 and Glen R Shaw1,3,5
Address: 1National Research Centre for Environmental Toxicology, University of Queensland, 39 Kessels Road, Coopers Plains, QLD 4108, Australia, 2School of Population Health, University of Queensland, Herston Road, Herston, QLD 4006, Australia, 3Cooperative Research Centre for Water Quality and Treatment, PMB 3, Salisbury, SA 5108, Australia, 4Faculty of Health and Environmental Sciences, Auckland University of Technology, Private Bag 92006, Auckland 1020, New Zealand and 5School of Public Health, Griffith University, University Drive, Meadowbrook, QLD 4131, Australia

Email: Ian Stewart* - [email protected]; Philip J Schluter - [email protected]; Glen R Shaw - [email protected]

* Corresponding author

AbstractCyanobacterial lipopolysaccharide/s (LPS) are frequently cited in the cyanobacteria literature astoxins responsible for a variety of heath effects in humans, from skin rashes to gastrointestinal,respiratory and allergic reactions. The attribution of toxic properties to cyanobacterial LPS datesfrom the 1970s, when it was thought that lipid A, the toxic moiety of LPS, was structurally andfunctionally conserved across all Gram-negative bacteria. However, more recent research hasshown that this is not the case, and lipid A structures are now known to be very different,expressing properties ranging from LPS agonists, through weak endotoxicity to LPS antagonists.Although cyanobacterial LPS is widely cited as a putative toxin, most of the small number of formalresearch reports describe cyanobacterial LPS as weakly toxic compared to LPS from theEnterobacteriaceae.

We systematically reviewed the literature on cyanobacterial LPS, and also examined the much lagerbody of literature relating to heterotrophic bacterial LPS and the atypical lipid A structures of somephotosynthetic bacteria. While the literature on the biological activity of heterotrophic bacterialLPS is overwhelmingly large and therefore difficult to review for the purposes of exclusion, wewere unable to find a convincing body of evidence to suggest that heterotrophic bacterial LPS, inthe absence of other virulence factors, is responsible for acute gastrointestinal, dermatological orallergic reactions via natural exposure routes in humans.

There is a danger that initial speculation about cyanobacterial LPS may evolve into orthodoxywithout basis in research findings. No cyanobacterial lipid A structures have been described andpublished to date, so a recommendation is made that cyanobacteriologists should not continue toattribute such a diverse range of clinical symptoms to cyanobacterial LPS without researchconfirmation.

IntroductionCyanobacterial LPS is attributed with a range of patholog-ical effects in humans, from gastro-intestinal illness, cuta-neous signs and symptoms, allergy, respiratory disease,

headache and fever. This review will present the studies ofcyanobacterial LPS, and will attempt to place the knowl-edge of these products within the broader understandingof LPS from Gram-negative heterotrophic bacteria. The

Published: 24 March 2006

Environmental Health: A Global Access Science Source 2006, 5:7 doi:10.1186/1476-069X-5-7

Received: 10 May 2005Accepted: 24 March 2006

This article is available from: http://www.ehjournal.net/content/5/1/7

© 2006 Stewart et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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paper will present an overview of the mechanisms of tox-icity of Gram-negative bacterial LPS, discussing the historyof its discovery and the present perception of its patho-genicity.

Cyanobacterial LPS and symptoms in humansTable 1 lists some of the signs and symptoms reportedlyassociated with exposure to cyanobacterial LPS, and refer-ences that imply particular symptoms or symptom groupsare explained by such exposures. Table 1 does not presentan exhaustive list of citations implicating cyanobacterialLPS with human illness. Many such references are foundin review articles, and the table does not include citationswhich discuss cyanobacterial LPS in the context of cyano-bacterial toxins without linking them to specific illnesses– e.g. "Cyanobacterial toxins are of three main types:lipopolysaccharide endotoxins, hepatotoxins, and neuro-toxins." [1]; "Potential irritant; affects any exposed tissue"[2,3]; "(LPS) are responsible for the irritant nature of

cyanobacterial material" [4]; and "...toxicants suchas...lipopolysaccharide endotoxins...affect any exposedtissue..." [5].

Several authors note that the health implications ofcyanobacterial LPS are poorly understood and the topicrequires more research [6-15]. Only one reference wasfound in the cyanobacteria literature that raises doubtsabout illness caused by cyanobacterial LPS: Carmichael[16] suggests that the relationship between ingested LPSand illness in an immunologically competent populationis debatable, there being little evidence that people with anormal LPS-containing gut flora would be affected by LPSfrom water supplies.

The relationship between cyanobacterial LPS and illness isdiscussed in language ranging from cautious: "...may beresponsible..." [17], "possibly (due to) lipopolysaccha-rides" [15], to definitive: "dermatotoxins" [18] and "der-

Table 1: Signs and symptoms attributed to contact with cyanobacterial lipopolysaccharides.

G-I Skin Eye Allergic Respiratory Hay-fever Headache Dizziness Cramps Blistering of mucous

membranes

Fever References

[168, 169, 217, 262-268]

[23, 269-273]

[6, 12, 274-276]

*[20, 277*-279]

[257]

**[11**, 280]

***[59, 261***]

[26, 281]

[17]

[282]

[19]

[15, 21, 283]

[206]

[284]

[285]

* "cytotoxic" skin irritation** "allergic and toxic responses"*** "respiratory allergy"



matotoxic lipopolysaccharides" [19,20]. CyanobacterialLPS has also been implicated as the cause of an outbreakof pyrogenic reactions in a haemodialysis clinic in 1974[21].

The references in the literature to the association betweencyanobacterial LPS and this rather diverse range of symp-toms are not based on any research evidence specific tocyanobacterial LPS. As will be discussed later in thisreview, the few toxicological investigations that have beencarried out to date are mostly limited to the end-points oflethality and the local Shwartzman reaction, in whichsequential subcutaneous and intravenous injections ofLPS produce a dermonecrotic lesion in rabbit skin. Rather,symptomatology attributed to contact with cyanobacterialLPS appears to be something of a default diagnosis for ill-nesses that are not otherwise explained by the currentknowledge of cyanobacterial exotoxins, most of which aresomewhat specific to their target organ system.

The most likely explanation for the ready attribution ofthese illnesses to cyanobacterial LPS lies in the realisationthat LPS from Gram-negative heterotrophic bacteria areimplicated in significant morbidity – and mortality – socyanobacteria, which are widely but somewhat inaccu-rately accepted as Gram-negative bacteria may equally beresponsible for illness because they contain LPS. Coddmakes such a proposition, suggesting that: "The LPS ofother bacteria are associated with gastroenteritis andinflammation problems and it is thought that cyanobac-terial LPS may contribute to waterborne health incidentsalthough this possibility has not been adequately investi-gated" [13].

There is a risk that speculative attribution of symptoms inhumans to environmental exposure to cyanobacterial LPSwill continue without an appropriate and specific researchfoundation. This review will attempt to examine moreclosely the mechanisms by which LPS from Gram-nega-tive heterotrophic bacteria is associated with morbidity,and compare and contrast cyanobacterial LPS and hetero-trophic bacterial LPS.

Cyanobacteria: Gram-negative or Gram-positive?Most references to cyanobacteria describe these organismsas Gram-negative prokaryotes [17,22-26]. Weckesser et al[27] state that the presence of LPS is evidence for theGram-negative cell wall architecture of cyanobacteria.However, Weckesser et al [28] reported that a strain ofAnabaena variabilis they investigated was Gram-positive,"like other blue-green algae...". Drews [29] also classifiescyanobacteria as Gram-positive. Golecki [30] reviewedelectron microscopy studies of the cell wall architecture ofvarious bacteria, and suggested that cyanobacteria havecharacteristics of both Gram-negative and Gram-positive

organisms. They contain an outer membrane and LPS,which are defining characteristics of Gram-negative bacte-ria [31,32], and a thick, highly cross-linked peptidoglycanlayer similar to Gram-positive organisms. Jürgens &Weckesser [33] and Golecki [30] suggest that the chemicaland structural organisation of the cell wall may placecyanobacteria in a separate phylogenetic category to bothGram-negative and Gram-positive bacteria, with cyano-bacteria developing independently of Gram-negative andGram-positive bacteria from a common ancestor. How-ever, according to Margulis & Schwartz [34], electronmicroscopy shows that cyanobacteria have Gram-negativecell walls.

Terminology: "endotoxin" in the cyanobacteria literatureWhile most references to endotoxin in the cyanobacterialiterature (e.g. those in Table 1) are clearly referring toLPS, i.e. cell wall structural components, this is not alwaysthe case. Several authors discuss "endotoxins" when theywere obviously describing the toxic properties of micro-cystins [35-41]. Kay [42] refers to aphantoxin (nowknown as saxitoxins) from Aphanizomenon flos-aquae as an"alkaloid endotoxin". Gentile & Maloney [43] alsolabelled what were presumably saxitoxins as an endo-toxin. In recent years cyanobacterial hepatotoxins andneurotoxins are still being described as endotoxins[44,45]. This misunderstanding of the nomenclature isalso seen in biotoxin research fields outside of cyanobac-teria, with brevetoxins described as endotoxins [46].Immunologists clearly understand endotoxins to referonly to LPS of Gram-negative bacteria. The reader's atten-tion is drawn to this distinction, as throughout this review"endotoxin" and "LPS" are used more or less interchange-ably, whereas other cyanobacterial toxins such as micro-cystins, cylindrospermopsin, saxitoxins and anatoxin-a,when discussed in aggregate are referred to as "exotoxins".The history of the term "endotoxin" and its current use arediscussed in the following section.

Gram-negative bacterial LPS: introduction and its discoveryLPS from many bacterial species will initiate acute inflam-matory responses in mammals that are typical of the hostreaction to tissue injury or infection [47]. LPS can inducea large and diverse range of effects, ranging from pyrexiato Gram-negative septic shock, which manifests as a com-plex and dramatic syndrome involving fever, leucopoenia,hypotension, cardiopulmonary dysfunction, dissemi-nated intravascular coagulation and multi-system failure[48,49].

The history of the understanding of LPS starts in the latenineteenth century, when Richard Pfeiffer used heat-inac-tivated lysates of Vibrio cholerae to provoke pathophysio-logical effects in guinea pigs [48]. Pfeiffer named the heat-



stable and cell-associated toxic substance "endotoxin", todistinguish it from the heat-labile and proteinaceous exo-toxins, which were known to be secreted by replicatingbacteria [48-51]. Initial analyses of endotoxin revealedthat it contained polysaccharide and lipid, and was thusnamed lipopolysaccharide. The terms "endotoxin" and"lipopolysaccharide" are widely used by workers in a vari-ety of biomedical fields as synonyms to describe the samemolecule (many references, e.g. [48,51-57] including thefield of cyanobacteria research: "lipopolysaccharide endo-toxins" [20,58-60]. However, some authors have sug-gested that LPS should refer to the purified molecule,whereas endotoxin more appropriately describes macro-molecular complexes of LPS, protein, phospholipid andnucleic acids [49,50,61].

The study of LPS, which was largely concerned with inves-tigating fever, progressed in the late 1940s with the discov-ery of endogenous pyrogens [49]. Since then, the field ofcytokine biology has made enormous strides. It is nowunderstood that the pathological effects of LPS are indi-rect, i.e. LPS acts by initiating a cascade of host-mediatedresponses: initially monocytes and macrophages are stim-ulated, and then neutrophils and platelets congregate inmicrocapillaries, causing vascular injury. Inflammatorycells release a range of endogenous mediators, includingarachidonic acid metabolites, platelet-activating factor,cytokines such as interleukin-1 (IL-1), IL-6 and tumournecrosis factor-alpha (TNF-α), nitric oxide, toxic O2metabolites, vasoactive amines, proteases and products ofthe complement and coagulation cascades. There aremany reviews in the literature on cytokines and LPS –some recent examples are: [47-49,51,55,56,62-64].Cytokine-mediated responses are extremely complex:cytokines are pleiotropic, i.e. a single type of cytokineaffects several different types of cell. Cytokines have auto-crine effects, i.e. they can stimulate the cells that secretethem to produce more cytokines, and paracrine effects –stimulating other cells to produce different cytokines [65].Signs and symptoms in human volunteers after adminis-tration of endotoxin are: fever, rigors, and influenza-likesymptoms of fatigue, headache, nausea, myalgia, arthral-gia, drowsiness, mild amnesia and diarrhoea[47,61,66,67].

From the discovery that LPS-associated pathology resultsfrom the stimulation of host cell responses came the real-isation that LPS binds to specific receptors in order toelicit the release of cytokines and other inflammatorymediators [64]. Several membrane-bound and solubleproteins have been shown to bind LPS; the most impor-tant appear to be CD14 and LPS-binding protein (LBP)[48,51,52,55,63,64,68]. The Toll-like receptor (TLR) fam-ily are a recently discovered group of transmembranereceptors that are fundamental in signalling innate

immune responses to conserved microbial structures, andthey are also involved in the recognition of some endog-enous ligands. TLR4 is instrumental in signalling LPSfrom many Gram-negative bacteria, and TLR2 is involvedin the recognition of some unusual LPS. There is a consid-erable and rapidly expanding body of literature on theTLR family; one of the most comprehensive reviews is byTakeda et al [69]. A review by Janeway and Medzhitov[70] is an excellent introduction to TLRs, and places theactivity of these receptors in the broader context of innateimmunity.

Knowledge of the role of specific receptors in mammalianhosts has led to the demonstration that LPS itself is non-toxic. Schnaitman notes that "LPS itself is not toxic" [71]and Henderson et al describe LPS as "relatively inactive"and observe that several host proteins are necessary forLPS to display full agonist potency [49]. CD14-deficientcell lines such as Chinese hamster ovary are unresponsiveto LPS at high doses, and cell lines that show poor LPSactivation can be converted to show high activation whentransfected with the CD14 gene [71]. LPS-unresponsivemouse strains and cytokine knockout strains also serve toreinforce the concept that LPS is not directly toxic, and thepathophysiology associated with Gram-negative LPSresults from host-mediated factors [68,72-77].

LPS structure – similarities and differences across Gram-negative bacteriaLPS from different Gram-negative species apparentlyshare common features in their basic architecture. A struc-ture consisting of four covalently linked segments – a sur-face carbohydrate polymer (O-specific chain), a coreoligosaccharide featuring an outer and inner region, andan acylated glycolipid (termed lipid A) – is seen in suchecologically diverse bacteria as Salmonella, Pseudomonas,Vibrio and Rhizobium [78]. The O-specific chain shows themost diversity, and is the basis for serological specificity[48], while lipid A, which anchors the LPS molecule in theGram-negative outer membrane, is the most conservedbiochemical structure across different bacterial species[72] (See Figure 1). There is unequivocal acceptance thatthe lipid A moiety is the innate immune stimulating or"endotoxic" component of LPS [63,79]. This was con-firmed by the observations that LPS from polysaccharide-deficient mutant strains were equally as bioactive as par-ent LPS [63,72,80], and chemically synthesised Escherichiacoli lipid A exhibits identical activity to natural E. coli lipidA [63,81].

Are all LPS equal in terms of their host-mediated responses?Although the basic structure of lipid A is seen in phyloge-netically diverse Gram-negative bacteria, variations in thenature and location of acyl groups or alterations in the



hydrophilic backbone can result in partial or total loss ofbiological activity [48,51,63,82,83]. An example is seen inthe case of some purple non-sulfur bacteria, which couldbe considered to be somewhat more representative ofcyanobacteria when it comes to drawing comparisons onthe biological activity of LPS. Purple non-sulfur bacteria,being photosynthetic, are Gram-negative bacteria thatoccupy a similar ecological niche to that of cyanobacteria,and therefore have growth and reproductive strategies thatmuch more closely resemble cyanobacteria than the het-erotrophic, gut-dwelling E. coli and Salmonella. Lipid Acomplexes from Rhodobacter sphaeroides and Rhodobactercapsulatus are not only inactive, but are antagonists ofEnterobacteriaceae LPS-induced cell activation[48,49,52,63,84]. R. sphaeroides and R. capsulatus lipid Adiffers from that of Escherichia coli in several respects, allrelating to the acylation pattern; the lack of endotoxicactivity in R. sphaeroides and R. capsulatus appears to beattributable to the presence of five rather than six fattyacid groups, and the shorter chain lengths (C10 in R.sphaeroides and R. capsulatus compared with C14 in E. coli)in two ester-linked fatty acids [84]. Lipid A from R. sphaer-oides and R. capsulatus is of pharmacological interestbecause of their LPS-antagonist properties; these lipid Astructures and some synthetic derivatives have beenshown to be potent LPS antagonists in vitro and to protectagainst LPS-induced morbidity and mortality in animalmodels [49,63,78,85]. Competitive inhibition of biologi-cally active LPS by LPS antagonists further illuminates therequirement for host cell receptor sites in mediating theresponses to LPS [52]. Other purple non-sulfur bacteria,e.g. Rhodopseudomonas viridis and Rhodopseudomonas palus-tris are also reported to lack endotoxic activity [27,84], butthe lipid A from another purple non-sulfur bacterium,Rubrivivax gelatinosus, is reportedly associated with highlethality in mice – at similar doses that cause lethality inSalmonella – and high pyrogenicity in rabbits [27,84]. The

high endotoxicity of R. gelatinosus is apparently due to thepresence of six fatty acid groups in the lipid A structure[84,86]. Lipid A complexes from the pathogenic bacteriaChlamydia trachomatis and Legionella pneumophila and thenon-pathogenic hyperthermophile Aquifex pyrophilusreportedly possess little or no LPS agonist or antagonistproperties [63].

Bacteroides spp., which are common gut and periodontalcommensals, have low endotoxic activity compared tothat of Salmonella, with a greater than 500-fold highermouse LD50 and 100 to 1,000-fold reduced ability to stim-ulate production of IL-1 in monocyte cultures [87,88].Bacteroides fragilis LPS inhibits E. coli LPS-inducedendothelial adhesiveness for polymorphonuclear leuco-cytes, although B. fragilis LPS is reported to be directlytoxic to endothelial cell cultures at high concentrations[52]. In a study of cytokine induction in whole blood, Fri-eling et al [89] showed that B. fragilis stimulated IL-1β, IL-6 and IL-1ra at levels comparable to Gram-positive bacte-ria, where 100 to 1,000-fold more organisms are requiredto produce similar concentrations of cytokines than withcommon pathogenic Gram-negative bacteria. Wilson et al[90] suggest that B. fragilis may be such a weak cytokineinducer because, being a dominant organism in normalgut flora, it has evolved mechanisms to downregulate thesynthesis of inflammatory cytokines in order to optimisetheir niche in the host gut. B. fragilis has five fatty acylgroups in the lipid A moiety, and other structural differ-ences to Salmonella and E. coli LPS, such as a monophos-phorylated disaccharide backbone and longer fatty acylchains [91].

Some pathogenic bacteria have LPS which reflects thehighly specific niches they inhabit: enteric Gram-negativebacteria have long, hydrophilic and neutral O-specificpolysaccharide chains which protects the organism fromsolubilisation by bile acids and intestinal enzymes,whereas organisms that colonise the mucous membranesof the respiratory and genital tracts have outer membranesurfaces that are hydrophobic and can be solubilised bybile [92,93]. Gram-negative bacteria such as Neisseria,Haemophilus and Bordetella have developed unique surfaceglycolipids lacking O-antigens, which some workers calllipooligosaccharides [63,92,93].

The presence of one or more secondary acyl chainsappears to be essential for lipid A to stimulate some endo-toxic reactions [82,88]. One of the final stages of Entero-bacteriaceae lipid A biosynthesis is the formation ofacyloxyacyl groups, so-called secondary fatty acids [94]. Aleucocyte enzyme, acyloxyacyl hydrolase, selectivelyremoves these secondary fatty acyl groups, without releas-ing the 3-hydroxy acyl chains that substitute the lipid Adisaccharide backbone [82,95]. Deacylated LPS from E.

Schematic of the basic LPS structureFigure 1Schematic of the basic LPS structure. The O-specific polysaccharide is the unit that is most exposed to the exter-nal environment and so manifests the greatest structural diversity; lipid A is the most conserved structure.



coli, Salmonella typhimurium, Haemophilus influenzae andNeisseria meningitidis were shown to have reduced activi-ties in a series of tests relating to endotoxic potential, insome cases by greater than two orders of magnitude, anddeacylated Neisseria LPS demonstrated some antagonisticactivity towards Neisseria and Salmonella LPS [82,95,96].

The lipid A analog, compound 406, which lacks the twosecondary fatty acids of E. coli lipid A, is unable to inducecytokines in human cells [48,51,75]. Rietschel et al [51]propose that endotoxic capacity resides in the spatial con-formation of lipid A, in that biologically inactive lipid A(e.g. R. capsulatus) conforms to lamellar structures,whereas endotoxic lipid A adopts exclusively cubic or hex-agonal structures.

Netea et al [97] suggest that the historical assumption thatLPS from different Gram-negative bacteria possess similarbiological effects is incorrect, and that differences betweenLPS across species are the rule rather than the exception.The authors discuss structure-function relationshipsbetween LPS and the Toll-like receptors (TLRs), which areintegral in LPS-mediated signalling, suggesting that differ-ences in the three-dimensional conformation of LPS mol-ecules translate into differences in TLR signalling ofproinflammatory cytokines. In their reviews of thesupramolecular structure and phase transition states ofLPS and lipid A, Seydel and Brandenburg [98] and Seydelet al [54] suggest that endotoxic activity is related to thethree-dimensional conformation of LPS and the mul-timeric aggregates they form. The conformation of LPS orlipid A within such aggregates is not constant, but a revers-ible phase transition occurs, which is temperature-dependent and related to the length and degree of satura-tion of the acyl chains and other physiological conditions.

More recent work by Seydel and collaborators has led tothe realisation that the biological activity of specific lipidA structures can be determined by an understanding oftheir supramolecular structure, which is a function of themonomeric conformation, which in turn is largely deter-mined by the primary molecular structure [83,99,100].The presence of sufficient negative charges on the disac-charide backbone – mainly, but not necessarily, two phos-phate groups – has an important influence on lipid Amolecular conformation and the binding capacity toserum proteins such as LBP [83,101]. A high negativecharge density is reported to be an essential requirementfor agonistic and antagonistic properties; complete or par-tial substitution of negative charges can result in the lossof all biological activity [83,102]. Seydel's team now sug-gest that a general principle can be applied regarding themolecular conformation of lipid A structures and theirbiological activity: lipid A structures that adopt conical/concave shapes have hexagonal or cubic supramolecular

aggregate structures and express high endotoxic potential,whereas lipid A structures that adopt cylindrical confor-mations have lamellar aggregates and are either inactiveor LPS antagonists [99]. Thus lipid A of the photosyn-thetic bacterium R. gelatinosus, which has high endotoxicactivity [27,84], was seen to adopt hexagonal aggregatestructures, whereas the lipid A formations of other non-endotoxic photosynthetic bacteria adopt cylindricalshapes and lamellar aggregate structures [99,100,103].Lipid A from Chromobacterium violaceum forms a cylindri-cal geometry and is reportedly three orders of magnitudeless active than E. coli lipid A [104]. Ulmer et al [105] alsofound that a synthetic lipid A structure based on C. viol-aceum lipid A had markedly lower cytokine-inducingcapacity than an E. coli-based synthetic lipid A. These find-ings are in contrast to some other reports, which describeC. violaceum lipid A as having a high agonist activity[51,75]. Of interest is the observation that the primarylipid A structures of C. violaceum and R. gelatinosus are verysimilar (bisphosphorylated diglucosamine backbone, sixsymmetrically distributed acyl chains differing only in thelength of the two acyloxyacyl groups: C = 12 in C. viol-aceum and C = 10 for R. gelatinosus) [84]. Yet C. violaceumlipid A is an LPS antagonist, whereas R. gelatinosus lipid Aexpresses high agonist activity. Seydel et al[104] suggestthat the chemical structures of these two lipid A com-plexes may need to be re-examined. Lipid A of Campylo-bacter jejuni, which has low endotoxic potential, shows avery slight tendency to adopt a conical/concave shape,whereas the lipid A of E. coli clearly adopts a conical/con-cave form [99,106].

As a result of these studies, the ability of a lipid A mono-mer to adopt a conical shape (the so-called endotoxic con-formation) has been described as a prerequisite forendotoxicity [100]. Seydel et al [104] suggest that whenlipid A molecules are intercalated into target cell mem-branes, only lipid A which forms a conical shape – wherethe cross-sectional area of the hydrophilic backbone issmaller than the cross section of the hydrophobic acylgroups – can exert a mechanical stress on signalling pro-teins. LPS with a lipid A moiety which assumes a cylindri-cal shape – cross-sectional areas of the hydrophobic andhydrophilic components being roughly equal – willoccupy the binding site but be unable to activate signal-ling proteins, thus acting as an LPS antagonist [102,104].The number and distribution of acyl chains has beenshown to affect the tilt angle of the disaccharide backbonewith respect to the target cell membrane; the orientationof the backbone sugars appears to correlate with the endo-toxic potential of the LPS. The lipid A molecular shapeand the tilt angle of the backbone sugars are reported tobe the complete determinants of endotoxic activity[102,104].




Primary lipid A structuresFigure 2Primary lipid A structures. E. coli has a bis-phosphorylated diglucosamine backbone with six amide and ester linked fatty acyl chains. The non-endotoxic (and LPS-antagonist) R. sphaeroides lipid A has an identical disaccharide backbone but has five acyl residues, shorter ester-linked primary acyl chains and an unsaturated acyl group. B. fragilis, which expresses low endotoxic potential, has a mono-phosphorylated backbone and five acyl chains with longer chain lengths than those seen in E. coli. Lipid A from Chromobacterium violaceum, like R. sphaeroides, is an LPS antagonist. Figures adapted from: Weintraub et al [91], Rietschel et al [78] and Takayama & Qureshi [84].


The discussion above has contrasted the endotoxic poten-tial of lipid A structures from the most widely studiedforms, those of the Enterobacteriaceae, with some unu-sual lipid A complexes from photosynthetic bacteria andsynthetic lipid A analogs. The reason that cyanobacterialLPS has not been discussed here is simply that therequired research has not been done as yet. No cyanobac-terial lipid A structures have been published, therefore noinferences can be deduced as to their likely endotoxicpotential, or lack of it. But with the knowledge that endo-toxic potential can vary in the most fundamental wayacross Gram-negative bacteria, from agonistic to weaklyactive to inactive to antagonistic, it should be incumbenton the cyanobacteria research community to cease attrib-uting biological activity and clinical symptoms to cyano-bacterial LPS without specific research evidence.Cyanobacteria may not be typical Gram-negative organ-isms because of their unusual cell wall architecture, andcyanobacteria will have experienced very different selec-tion pressures to gut-dwelling Gram-negative bacteria,which may be reflected in different lipid A structures.

LPS and infectionMuch research into LPS and lipid A has understandablyconcentrated on the severe, life-threatening hostresponses to circulating LPS that constitute Gram-negativesepticaemia and septic shock. Many experimental modelshave utilised either in vitro studies of isolated cell lines oranimal and human studies where LPS is exposed to thecirculation by parenteral injection.

The matter of the degree to which cyanobacterial LPS/lipid A can stimulate mammalian cytokine networksunder experimental conditions, which remains largelyunanswered, is only one aspect in the understanding ofcyanobacterial LPS and the gastro-intestinal, respiratoryand cutaneous illnesses which have been attributed tocontact with it. The other side of the story that needs to beconsidered is the mechanism by which cyanobacterial LPSmight (and might not) stimulate endotoxic responses bythe various natural exposure routes: ingestion, inhalationand contact. The following discussion will briefly reviewthe association between acute gastrointestinal illness andGram-negative bacterial LPS. Cutaneous responses to LPSwill be briefly discussed.

Terminology: infection, pathogenInfectious diseases caused by bacteria are characterised byseveral discrete steps: bacterial adhesion to the host, colo-nisation within or on the host, and evasion of hostdefences [31,49]. The only references in the literature thatdescribe cyanobacteria as invasive, infectious organismswere from two authors:

• Rank [107-109] put forward the hypothesis that achronic, low-virulence infestation by cyanobacteria grow-ing heterotrophically may explain the aetiology of arterio-sclerosis in humans and homeothermic animals. Theauthor bases his theory on ecological data, examining thegeographical, demographic and historical distribution ofthe disease. Routes of infection are posited to be ingestionof unfermented milk, public water supplies drawn fromsurface waters as distinct from groundwater [108,109],and earth-contaminated food and other objects [107].The author also critiques the three theories of the patho-genesis of arteriosclerosis that were current at the time(response-to-injury, lipid hypothesis, and monoclonalhypothesis) and suggests laboratory studies to replicateand study the disease. Such studies do not subsequentlyappear in the literature, and no further reference to thecyanobacterial infection hypothesis can be found.

• Ahluwalia et al [110] and Ahluwalia [111,112] positedthe theory that Microcystis aeruginosa is the causativeorganism of the mostly tropical, water-exposure-relatedinvasive disease rhinosporidiosis. However, this theoryhas been disputed; convincing and probably conclusiveevidence appears to place the eukaryotic protist Rhinos-piridium seeberi as the causative organism (see Author'sreply to Ahluwalia [112]).

Most public health workers would presumably placecyanobacteria-related illnesses in the context of environ-mental exposures, as distinct from familiar diseases due tocommunicable infectious bacteria, which in many casesfeature transmissible illnesses, secondary to the originalreservoir of infection. Cyanobacteria-related illness isviewed as intoxication, rather than infection, usually onthe basis of a sudden onset of symptoms occurring soonafter exposure (i.e. without an incubation period), andlack of secondary cases [113]. Giesecke [114] definesinfectious disease as "all diseases caused by micro-organ-isms", with sub-definitions of communicable and trans-missible disease (communicable disease: capable of beingtransmitted from an infected individual to another per-son, directly or indirectly; transmissible disease: able to betransmitted from one individual to another by 'unnatural'routes). Whether cyanobacteriologists would embracethat definition of infectious disease is debatable, but mostwould agree that cyanobacteria-related diseases are nei-ther communicable nor transmissible. Exotoxin-produc-ing cyanobacteria certainly fit the dictionary definition ofpathogenic (i.e. disease-causing) organisms; the reader'sattention is drawn to the distinction between infectious(implying colonisation and evasion of host defences)Gram-negative bacteria and non-infectious cyanobacteriain the following discussions on LPS and G-I and dermalillnesses.



Mechanisms of vomiting and its relationship to LPS activityNausea and vomiting are normal physiological responsesto the ingestion of toxic substances; they are essentialdefences because they are the end result of the actions ofsensorimotor systems that operate to identify and rapidlyexpel hazardous substances from the upper G-I tract [115-117]. The two main sensory systems that direct the emeticresponse are local, associated with the gut mucosa (pre-absorptive response), and central, specifically the chem-oreceptive trigger zone of the area postrema, located in thedorsal surface of the medulla oblongata (post-absorptiveresponse) [118-120]. Stimulation of chemoreceptors inthe stomach, jejunum and ileum by irritant chemicalssuch as hypertonic saline, copper sulfate or mustard, or bybacterial enterotoxins, leads to the activation of vagal sen-sory afferent nerves to the brain. Vagal efferent processingthrough the enteric nervous system stimulates entericmotor neurons to effect emesis [115,117,119,120].Emetic chemoreceptors are also found in the vascular sys-tem; activation of these chemoreceptors will also initiatenausea and vomiting [115]. Endogenous mediators ofemesis such as dopamine, acetylcholine and enkephalinare reported [115]. Prostaglandins have well-knownemetic actions [121].

Circulating E. coli LPS is a potent emetic stimulant. In aseries of experiments using piglets, Girod et al [122]showed that parenteral administration of LPS provokedvomiting (as well as fever, rigors, purpura, diarrhoea anddrowsiness). The authors suggested that LPS stimulatesvomiting by means of cytokine-induced prostaglandinsand other endogenous mediators acting both centrallyand on vagal afferents. Other animal studies have demon-strated the emetic action of LPS [123].

The area postrema is reported to be the primary sensoryarea involved in nausea as well as vomiting, although nau-sea is accompanied by autonomic excitation, whereasvomiting is a somatic process independent of the auto-nomic nervous system [118,121]. Presumably LPS-stimu-lated endogenous mediators are associated withsymptoms of nausea, which is reported in several studiesand reviews of intravenous exposure to LPS in human vol-unteers [47,61,66,67].

To investigate vomiting associated with exposure tocyanobacteria, the appropriate research efforts shoulddetermine whether cyanobacterial LPS is capable of stim-ulating gut chemoreceptors, or if cyanobacterial LPS cangain access to the circulation and stimulate nausea andvomiting centrally. Another point of interest should bedirected towards the cyanobacterial exotoxins, as to theircapacity to stimulate either gut mucosal chemoreceptors,vascular emetic chemoreceptors, or whether they inducevomiting through the activity of endogenous mediators.

Bacterial exotoxins such as staphylococcal enterotoxins(SEs) are known to stimulate emesis via gut chemorecep-tors [116,117,124]. Worthy of note is that experimentalinduction of LPS-related emesis is achieved by intrave-nous or intraperitoneal routes [122-127], whereas SEsreadily elicit vomiting when administered intragastrically[124,128]. Also of interest with respect to cyanobacteria-related G-I illness are some similarities of clinical symp-toms in the case of staphylococcal food poisoning: enteri-tis due to ingestion of SEs is characterised by rapid onset(1–4 hours) of vomiting ± nausea and diarrhoea, abdom-inal cramping and dizziness [124,129]. Staphylococcalfood poisoning is an intoxication, not an infectious proc-ess [129].

Diarrhoea and LPSOne inference that can be drawn from the references thatposit cyanobacterial LPS as the cause of G-I illnesses (seeTable 1) is that these symptoms are not necessarily relatedto exposure to any of the known cyanobacterial exotoxins,the assumption being that exposure to – and presumablyillness caused by – cyanobacterial LPS can occur with orwithout concurrent exposure to exotoxins (i.e. from non-toxic strains or species, or non-production of exotoxins atthe time of exposure). However, several questions need tobe answered if LPS is the sole presumptive "G-I-toxin". Ahypothesis is needed for the mechanism of cyanobacterialLPS, in the absence of other virulence factors, to initiatediarrhoea by the oral exposure route. Another explanationfor cyanobacterial LPS-related diarrhoea is that the altera-tion in gut membrane permeability may be related to acytokine cascade generated by circulating cyanobacterialLPS, but again, a hypothesis is needed to explain the expo-sure route for the LPS to overcome innate immune intes-tinal defences in order to gain access to the circulation.

Some observations on the behaviour of Gram-negativebacterial LPS in the gut serve to cast doubt on the suspi-cions that cyanobacterial LPS alone is responsible for ini-tiating acute gastro-intestinal illness in humans by theoral route:

• Commensal gut flora: The human intestinal tracthouses an enormous population of bacteria, many ofwhich are Gram-negative. The Enterobacteriaceae arefound in normal faecal flora at some 108–109 per gram[130]. The number of microbes in the gut lumen exceedsthe number of eukaryotic cells in the human body by anorder of magnitude [49,131], an observation that maylead some to unkindly suggest that the principal reasonfor human existence is to serve as bags for the housing andtransport of bacteria. Nanthakumar et al [132] note thatmature enterocytes are 100 to 1,000 times less sensitive toLPS than neutrophils and hepatocytes, which is not sur-prising since they are exposed to Gram-negative bacteria



and their endotoxins since birth when the gut is colo-nised.

• Non-virulent strains: Most Gram-negative organismsare non-pathogenic. Pathogenicity involves a complexinteraction between host-related and specific microbialvirulence factors – the latter including pili, fimbriae andheat shock proteins [133,134]. Infectious, i.e. colonising,microbes are the most common cause of diarrhoea world-wide; pathogenic strains commonly cause disease by theaction of enterotoxins [135]. That virulence factors otherthan lipid A structures of LPS are responsible for gastro-intestinal disease is seen in the protective effects of atten-uated or mutant Gram-negative bacteria when used as liveoral vaccines against pathogenic strains [133,136-138].Some E. coli strains are used as probiotics for the treatmentof gastrointestinal disease and infection prophylaxis inneonates [139].

• Anecdotal reports of consumption of non-hazardouscyanobacteria: Heaney [39] reports observations of cattleseen drinking from two Irish lakes affected by thick scumsof Anabaena flos-aquae and Aphanizomenon flos-aquae with-out ill effect. Author IS can add a similar observation: dur-ing recruitment for an epidemiology study [140] at LakeCoolmunda in southern Queensland, a frank Microcystisaeruginosa bloom was in attendance. A group of six orseven dogs were seen playing vigorously in the water, andthree dogs were observed drinking from it. The owners ofthe animals were questioned the following day; all deniedobserving any adverse effects. The consumption of Spir-ulina and other cyanobacteria provides further evidencethat cyanobacterial LPS cannot all be harmful. Cyanobac-teria as food, medicine and livestock feed will be dis-cussed later in this review.

Oketani et al [141] state that orally administered LPS isnot harmful to animals, which is in stark contrast to LPSadministered parenterally. Evidence for harmful effects oforally administered LPS is difficult to gather from the lit-erature because of the overwhelming number of publica-tions describing experimental use of LPS as an in vitro andin vivo immune stimulant. We found two reports of in vivoLPS activity by the oral route: Yang et al [142] showed thatoral administration of E. coli LPS enhanced the progres-sion of hepatoma in rats treated with thioacetamide.Yoshino et al [143], using a mouse model of autoimmunedisease, demonstrated that orally-dosed LPS exacerbatedcollagen-induced arthritis. These models of liver diseaseand autoimmunity are not applicable to the concept ofacute G-I symptoms caused by cyanobacterial LPS in pre-sumably healthy people in recreational settings andthrough cyanobacterial contamination of drinking watersupplies. However, they highlight the importance of gutmucosal immunity. LPS and other microbial products are

constantly sampled by gut-associated lymphoid tissues,which contain the largest assemblage of immunocompe-tent cells in the body [131]. Translocation of smallamounts of LPS from the gut lumen across the epitheliumand into the portal circulation is an important immune-stimulating process, with Kupffer cells in the liver playingan important role in clearance of LPS from the circulation[131]. LPS and other bacterial products from the normalgut flora can be a source of infection and sepsis when theintegrity of the gut mucosa is disrupted. This occurs in avariety of disease states, including hypovolaemic shock,burn injury, trauma, acute liver failure, pancreatitis, cir-rhosis and inflammatory bowel disease [131].

Roth et al [144] suggest that endogenous (i.e. gut-derived)LPS is a potent synergist of the toxicity of a range of struc-turally and functionally unrelated hepatotoxic xenobioticagents. The authors put the proposition that hepatotoxic-ity associated with some chemicals is indirectly caused byprimary damage to the intestinal tract, which allowsincreased translocation of bacterial LPS into the portal cir-culation. The liver is then exposed to harmful levels ofLPS, and the ensuing liver injury resembles that caused bylarge doses of LPS: changes in sinusoidal and parenchy-mal cells, neutrophil and platelet accumulation in sinu-soids, then multifocal hepatocellular degeneration andnecrosis. Yee et al[145] showed that co-administration ofindividually non-toxic doses of LPS and the alkaloid phy-totoxin monocrotaline produced significant liver injury,characterised by midzonal and centrilobular apoptoticand necrotic changes, coagulation and congestion, andloss of sinusoidal architecture. Ganey & Roth [146] pro-pose that the activation and increased expression of vari-ous soluble mediators, signalling molecules and cellularprocesses are crucial events in the augmentation of xeno-biotic toxicity by bacterial LPS, and that the mechanismsof toxicity are complex and variable.

Purified microcystins as well as microcystin-producingand cylindrospermopsin-producing cyanobacteria havebeen shown in accidental poisonings and through in vivoand in vitro experimental work to damage the gastrointes-tinal tract [37,147-153]. The possibility should be consid-ered that cyanotoxins and LPS (from cyanobacteria and/or from gut-dwelling heterotrophic bacteria) may cross adisrupted gut mucosal barrier and enhance the patholog-ical effects of cyanobacterial exotoxins.

LPS and cutaneous reactionsAs with LPS and oral exposure, searching bibliographicdatabases for evidence of LPS/endotoxin as the primarycause of acute clinical dermatoses is a difficult task, againbecause of the many citations of in vitro immunologywork using LPS to investigate various dermal cell proc-esses. As a starting point, dermatology textbooks were



perused, yet only one of a dozen or so standard texts madeany reference to either LPS or endotoxin: Rietschel &Fowler [154] describe a single case report of a hospitalworker who suffered dyshidrosis (a vesicular or vesicopus-tular eruption on the palms of the hands), which waslinked to endotoxin in latex gloves. Various dermatosesare clearly associated with either superficial or systemicinfection by many Gram-negative organisms, most nota-bly Pseudomonas aeruginosa [155]. However, it is unreason-able to compare mechanisms of cutaneous disease fromcolonising Gram-negative bacteria to those due to cyano-bacteria solely on the basis that both organisms containLPS.

Cyanobacterial LPSLiterature searches using PubMed and Web of Sciencewith the search terms (cyanobacteria* OR blue greenalga*) AND (LPS OR endotoxin) revealed 17 publicationsthat describe the extraction ± purification of cyanobacte-rial LPS. The Westphal hot phenol/water method wasused in 15 of these studies (described in [156]). Jürgens etal [157] used a sucrose density centrifugation and TritonX-100 extraction, and Papageorgiou et al [158] comparedthe phenol/water method with novel extraction methods.Raziuddin et al [159] used chloroform and acetic acidextraction to isolate lipid A from their LPS extract. Table 2lists the studies in which cyanobacterial LPS were testedfor lethality in mice. All doses were reported in the origi-nal papers in terms of dose per mouse; these doses havebeen converted to mg/kg body weight for comparisonpurposes in Table 2. An assumed weight of 20 g wasapplied to mice for the studies in which the authors didnot report the weight of their animals. Note that the studyof Scholtissek et al [160] used galactosamine-sensitisedmice, so their LPS doses are not directly comparable tothose given in the other studies; a similar situation applieswith the study of Katz et al [161], where adrenalectomisedmice were used. Adrenalectomy sensitises mice by threeorders of magnitude to the lethal effects of LPS [162,163].By way of comparison, some examples of reported lethalconcentrations of Enterobacteriaceae LPS are: 5–20 mg/kg[164], 6 mg/kg [165] and 24 mg/kg [163] (LD50 concen-trations in various strains of LPS-sensitive mice; assumedweight of mice = 20 g) to LD80 concentrations of 10–23mg/kg [166].

In addition to lethality, various other toxicity endpointswere examined in some of the studies listed in Table 2 andothers. Buttke & Ingram [167], Keleti et al [168] and Keleti& Sykora [169] investigated the local Shwartzman reac-tion, which is a dermonecrotic lesion elicited in rabbits bysubcutaneous preparative and intravenous provocationinjections. Positive Shwartzman reactions were generatedby Agmenellum quadruplicatum LPS, Schizothrix calcicolaLPS and Anabaena flos-aquae LPS, but not by Oscillatoria

tenuis LPS [167-169]. These results should be regarded asqualitative, as no reports were made of quantitative meas-urements of lesions, which can be done by various meth-ods [170,171]. However, the local Shwartzman responseto A. quadruplicatum LPS led the study authors to describeAgmenellum LPS as "a less effective endotoxin than Salmo-nella LPS" [167]. The Shwartzman reaction to A. flos-aquaeLPS was reportedly "weakly positive" [169]. Keleti &Sykora [169] also used a rabbit isolated ileal loop assay,which did not show any positive findings from the threecyanobacterial LPS isolates injected. Weise et al [172]tested A. nidulans LPS for pyrogenicity in rabbits, reportinga tenfold lower response than that seen from E. coli LPS.Schmidt et al [173] also tested the LPS from two Synechoc-occus strains for pyrogenicity, with maximum increases inrabbit body temperature of 1.5°C after injection of up to1 mg/kg. The authors reportthat these doses are somethree orders of magnitude higher than those required ofSalmonella LPS to achieve the same effect. LPS extractedand purified by author IS [156] and also by Papageorgiouet al [158] were investigated for their potential to affectthermoregulation in a mouse model. LPS from severalcyanobacterial isolates induced sickness behaviour andtransient hypothermia at doses of 70 mg/kg i.p.; similarresponses in positive control animals were seen after i.p.injection of 4 mg/kg E. coli LPS. Cyanobacterial LPS dosesof 7 mg/kg also initiated transient hypothermia and sick-ness behaviour, although at markedly lower intensity andduration than was seen with E. coli LPS at 4 mg/kg [156].

Most workers concluded that the cyanobacterial LPS theyexamined were weakly toxic when compared to the activ-ity of positive control heterotrophic bacterial LPS. Theexception was the work of Best et al [174], who investi-gated the potential of isolated cyanobacterial LPS toreduce the activity of glutathione S-transferases (GSTs) inZebra fish embryos. They reported that cyanobacterial LPSreduced microsomal and soluble GSTs in vivo to a greaterextent than LPS from E. coli or Salmonella typhimurium. Theauthors suggested that such reduction in GST availabilitymay deleteriously affect the ability of organisms todetoxify microcystins, presumably through decreased uti-lisation of glutathione (GSH) for conjugation reactions.This would seem to be a reasonable assumption, althoughother interpretations of this finding are possible. Whilethe GSH system is an essential intracellular redox buffer,preventing oxidative injury, it is also an important partic-ipant in many cellular functions, including DNA and pro-tein synthesis, metabolism, cell growth and amino acidtransport [175,176]. Many enzymes are GSH-dependent,including glutathione transferases [177]. GSH is also anessential component of some immune functions such asapoptosis, T-lymphocyte signalling and proliferation, andactivation of the nuclear transcription factor NFκB[176,178-184]. It is these immunological functions of the



GSH system that present as somewhat paradoxical: glu-tathione depletion has a protective effect on various mod-els of apoptotic and necrotic liver injury [181,185-187].Glutathione depletion has also been shown to preventLPS-induced inflammatory lung injury by attenuatingneutrophil activation and sequestration [188,189]. Glu-tathione depletion has been proposed as a novel anti-inflammatory pharmacotherapy [186,190]. Dröge et alwrote in 1994 that:

"It is clear that GSH is one of the limiting factors thatdetermine the magnitude of immunological functions invitro and in vivo...we must be prepared to reevaluate oneof the central dogmas concerning the role of GSH. GSHwas viewed mostly as an important anti-oxidant that pro-tects cells against oxidative stress" [178].

So the findings of Best et al [174], where cyanobacterialLPS markedly reduced GSTs in fish embryos after 24 hourexposures, may indicate an anti-inflammatory responsedisplaying a different time course to that seen with heter-otrophic bacterial LPS, or a consequence of decreasedGSH availability, as intracellular GSH efflux increaseswith the onset of apoptosis [179]. GSTs can be inhibitedby GSH conjugates, i.e. their reaction products [191], sothis possibility may also need to be considered. In anyevent, and insofar as innate immune responses in fishembryos can be equated to mammalian cellular activities,these responses are likely to be very complex, with numer-ous endogenous mediators, feedback loops and time-dependent variation. As an example, the chemoprotecantoltipraz, which is thought to exert anticarcinogenic effectsthrough induction of cytochrome P450 (CYP) enzymes, isnow known to transiently (within 24 hours) inhibit some

important CYPs and GSTs in vitro and in vivo. Animalstreated for three days showed two-fold-plus increases inthe same enzymes [192,193]. We would be interested tosee time-course studies that replicate the work of Best et al[174], perhaps continued over several days.

In addition to the studies cited above, nine reportsdescribe the isolation and purification of cyanobacterialLPS for various biomedical, biochemical or structuralstudies, but no toxicological endpoints were investigated[28,157,194-200]. The topic of cyanobacterial LPS wasreviewed by Weckesser et al [27], and Mayer & Weckesser[201]. These reviews are especially valuable in that theycontrast the work done on other photosynthetic prokary-otes, especially the purple non-sulfur bacteria, some ofwhich have been shown to have lipid A structures that areLPS antagonists, as discussed above. No cyanobacteriallipid A structures have been described to date.

In addition to work done on isolated and purified LPS,some reports discuss the activity of cyanobacterial LPS byuse of the Limulus amoebocyte (LAL) assay. This is ahighly sensitive test, though there are trade-offs in termsof specificity, with other bacterial products such as pepti-doglycan and glucan capable of registering positiveresponses [49,202,203]. The assay may not always be areliable predictor of cellular and in vivo responses [204].Seydel et al [205] suggest that, while the LAL assay is use-ful for detecting and quantifying LPS in blood products, itis not a measure of endotoxicity. The study of Rapala et al[206] had some significant findings pertaining to thetopic of presumed toxicity of cyanobacterial LPS. Theauthors used the LAL assay to test 26 axenic strains fromfive cyanobacterial genera; all responses were at least five

Table 2: Cyanobacterial lipopolysaccharides and lethality

CYANOBACTERIUM LETHALITY REFERENCE

Anacystis nidulans non-toxic at 10 mg/kg** [172]A. nidulans KM 2.5 mg/kg† (= approx. 800-fold greater than Salmonella minnesota LPS) [161]Phormidium spp. (x3) all non-toxic at mean dose of 333 mg/kg [286]Schizothrix calcicola non-toxic at 200 mg/kg [168]Anabaena flos-aquae UTEX 1444Anabaena cylindrica UTEX 1611Oscillatoria brevis

non-toxic at 250 mg/kgLD50 130 mg/kgLD50 190 mg/kg

[169][169][169]

Microcystis aeruginosa 006lipid AM. aeruginosa NRC-1lipid A

LD50 approx 45 mg/kg**LD50 60 mg/kg**LD50 40 mg/kg**LD50 approx 45 mg/kg**

[159][159][159][159]

Spirulina platensis Lb 1475/4a LD100 425 mg/kg** [223]Microcystis sp. PCC 7806 1 of 3 mice* died at 50 µg/kg** [160]M. aeruginosa (bloom sample)Cylindrospermopsis raciborskii AWT 205

no deaths at 70 mg/kgno deaths at 70 mg/kg

[156][156]

*galactosamine-sensitised (= TNF-α hypersensitised)† adrenalectomised mice** assumed weight of mice: 20 g



orders of magnitude lower than reactions to E. coli LPS,and several were below the assay's detection limits. Thissuggests that the lipid A structures of these LPS have somesignificant and fundamental structural differences toendotoxic lipid A, as the LAL assay does not react to someunusual or modified lipid A structures [88].

Spirulina platensis: the importance of exposure routeS. platensis has a long history of use as a foodstuff, dietarysupplement and livestock feed additive, with its probableuse dating back to the ninth century in Africa, and the 14th

century in Mexico [207]. Spirulina is classified taxonomi-cally under the genus Arthrospira, order Oscillatoriales; A.maxima and A. fusiformis are grown commercially in massculture, but usually designated as "S. platensis" [208]. Theuse of this cyanobacterium was comprehensivelyreviewed by Ciferri [209], who concluded that extensivenutritional and toxicological testing has shown it to be asafe and valuable protein source. The use of Spirulina wasbriefly reviewed along with that of other edible micro-algae by Kay [42], who cited Ciferri [209] in stating thatsome "negative effects" of Spirulina feeding were seen inmultigenerational studies and mutagenicity tests. How-ever, this appears to be a misinterpretation on the part ofKay [42], as Ciferri [209] described "negative results" fromthese studies. The original publications cited by Ciferri[209] were unobtainable. More recent studies seem tosupport the suggestion that consumption of Spirulina isnot harmful, and enhances various immune functions[210-214].

A single case report is the exception to the rest of the liter-ature. Iwasa et al [215] describe a 52-year-old male takingantihypertensive, hypolipidaemic and hypoglycaemicpharmacotherapy, who developed abnormal hepaticenzyme levels two weeks after taking Spirulina, presuma-bly on a regular basis. Liver function tests showed a signif-icant deterioration over the following three weeks, afterwhich he was hospitalised. Although a physical examina-tion was unremarkable, a liver biopsy revealed somedegenerative changes. Serological studies for a range ofviruses were negative. His medications and Spirulina werewithdrawn, after which his hepatic function rapidlyreturned to normal. Hepatotoxicity in this case was attrib-uted to consumption of Spirulina, on the basis of temporalrelationships between liver function abnormality andrecovery with consumption and withdrawal of Spirulina,although possible interaction effects with the medicationswould have been worth considering. This may have beenthe case with simvastatin, the cholesterol-lowering agentthis individual was taking. Simvastatin causes increasedproinflammatory cytokine production, and it can potenti-ate inflammatory responses induced by bacterial products[216]. A brief anecdotal report described two separateoccurrences of gastro-intestinal illness in adults following

consumption of Spirulina pills in Canada in the early1980s, though there was an indication that one individ-ual's tablets had some bacterial contamination [217].

Recent work has demonstrated that A. fusiformis in Kenyansoda lakes is capable of producing the cyanobacterial exo-toxins microcystin-YR and anatoxin-a [218,219]. Theimplications of this finding are important because cyano-bacterial poisoning is implicated in mass mortalities ofLesser Flamingos in the Rift Valley, and A. fusiformis is theprincipal food source for these animals [220,221]. Com-mon toxigenic cyanobacteria such as Anabaena and Micro-cystis are also found in these lakes, and periodicallydominate the phytoplankton profile [222], so presumablytoxin-producing genes are being transferred between thesegenera in the field.

The long-standing and widespread consumption ofArthrospira spp. illuminates the importance of consideringthe route of exposure in toxicology studies, and the dan-gers in this case of presumptive inference of disease fromthe findings of parenterally administered LPS. Tornabeneet al [223] reported a lethal dose of Spirulina platensis LPSin the range of 400 mg/kg (i.p. mouse), although thosefindings are not supported by the work of Stewart, whereSpirulina LPS i.p. injections of 350 mg/kg failed to induceeither thermoregulatory change or sickness behavioursigns [156]. However, Falconer [224] reported that celllysates of Spirulina were highly toxic to mice when admin-istered by intraperitoneal injection.

Other cyanobacteria are consumed as foods, medicinesand dietary supplements. Wild-harvested Aphanizomenonflos-aquae was over a decade ago reportedly "consumed bythousands of people without incident" [42], although thelake that produces the commercially available product(Lake Klamath, Oregon) is sometimes subject to contam-inating growth of Microcystis spp., and some A. flos-aquaeend-product batches contaminated with microcystinshave since been found [225-227]. A Canadian survey ana-lysed for microcystins in cyanobacterial products (Aphani-zomenon, Spirulina and unidentified cyanobacteria) butdid not present the results according to the componentgenera, so it is not clear whether Spirulina products (pre-sumably originating from commercial mass cultures) con-tained microcystins [228]. Nostoc commune, a terrestrialcyanobacterium, has a long history of use in China andScandinavia as food and medicine [229]. The widespreaduse of these products serves as a reminder that somecyanobacteria, and therefore their LPS, are not harmful bythe oral route.

LPS by inhalationIn our opinion, the sole natural exposure route that mightexplain aquatic cyanobacterial LPS-related illness is via



inhalation of aerosolised cells or fragments. Extrapolatingfrom the understanding of Gram-negative bacterial LPSon the respiratory system (as have most if not all of theauthors cited in Table 1 for the presumed involvement ofcyanobacterial LPS on various disease states), there is asignificant and increasing body of literature on the associ-ation between endotoxin and pulmonary disease, includ-ing asthma, chronic obstructive airway disease andemphysema. Intact bacteria and cell wall fragments arereadily aerosolised; bioaerosols of Gram-negative bacteriaare widespread contaminants of soils, water and livingorganisms [230,231]. Exposure to airborne endotoxin hasbeen associated with a range of occupational respiratorydiseases, in industries where high concentrations oforganic dusts are liberated, e.g. various agricultural set-tings, cotton milling, brewing, waste processing[230,232]. Endotoxin is also found in high concentra-tions in air pollution and household dust [233]. Endo-toxin in some aquatic environments can be aerosolised todisease-related concentrations: Rose et al [234] investi-gated outbreaks of granulomatous pneumonitis affectinglifeguards at an indoor swimming pool, with someaffected chronically. Gram-negative bacteria, principallyPseudomonas spp., colonised water spray systems in thefacility, and increased endotoxin in bio-aerosols waslinked to the illnesses.

Michel [232] reviewed experimental inhalation studies ofLPS: 4–12 hour periods of dyspnoea, chest tightness,myalgia, shivering, fatigue and malaise with or withoutfever were reported in a minority of normal subjects.Impaired pulmonary function in the form of bronchocon-striction, changes in non-specific bronchial hyperrespon-siveness and reduced alveolar-capillary diffusion weredemonstrated. Asthmatic subjects responded with signifi-cant bronchoconstriction lasting five or more hours atdoses of 20 µg, whereas normal subjects required doses of80 µg or more to produce moderate bronchoconstriction[232]. Of interest is the observation that LPS-induced lungchanges are associated with neutrophil activation,whereas purified allergen extracts induce bronchial eosi-nophilia in asthmatic subjects [232,235]. Normal subjectsexhibit a broad range of responses to inhaled LPS: 9% ofsubjects developed airway obstruction after low-doseinhalation, and 15% showed a negligible airway responseto high doses of LPS [233,236]. Polymorphisms in genescoding for Toll-like receptors, especially Toll-like receptor-4, appear to be important determinants of variability inhuman responses to inhaled endotoxins. Arbour et al[237] showed that a TLR4 sequence mutation is associatedwith an endotoxin hyporesponsive phenotype in humans.

Schwartz [233] describes asthma as a complex, heteroge-neous disease with multiple clinical sub-types, polygenicinheritance, and influenced by many different environ-

mental exposures. Endotoxin is one such exposure, whichcauses a biologically unique form of asthma [233]. How-ever, exposure to endotoxin early in life may confer bene-ficial effects: growing up on a farm and exposure tolivestock is reportedly associated with a significant reduc-tion in atopy, and there is an inverse correlation betweenhouse-dust endotoxin concentration and allergen sensiti-sation [238-240]. This so-called "hygiene hypothesis" forallergic diseases describes the concept that allergy resultsfrom an imbalance in the T-helper cell (Th) subset.According to this theory, exposure to bacterial and viralpathogens in the prenatal and early childhood periodsprevents the induction of allergen-associated Th2 cells byestablishing a Th1-biased immunity [238,241]. However,the hygiene hypothesis is complex and controversial, withcontradictory observations and refinements to the theoryappearing in the literature. Interested readers are directedto some recent reviews and updates: [241-244]

LPS and allergens initiate inflammatory processes in theairways through different pathways and cytokine cas-cades: LPS is recognised by innate immune cells, princi-pally alveolar macrophages, which generate pro-inflammatory cytokines such as IL-1, TNF-α and IL-8; thelatter cytokine recruits and activates neutrophils. LPS alsogenerates IL-12, which inhibits IgE responses. Allergensgenerate IL-4, IL-13 and IL-5, the latter cytokine being anactivator of eosinophils [241].

In the context of environmental exposures, endotoxinsand allergens often occur together; synergistic effects areimportant considerations in that airway responses tocombinations of LPS and allergen are reportedly greaterthan to either substance alone in atopic asthmatics[241,245].

The impact of cyanobacteria on respiratory symptoms inatopic individuals is worthy of investigation, and mayinvolve protein allergens and cyanobacterial endotoxinfrom both toxic and non-toxic blooms. However, the rel-ative burden of cyanobacterial endotoxin to respiratorymorbidity will depend on the capacity of the LPS of anygiven cyanobacterial species to act as an LPS agonist, or asan LPS antagonist, or be biologically inactive; such prop-erties are as yet largely undetermined.

An equally important research effort should be directedtowards the capacity of inhaled cyanobacterial exotoxinsto generate immunologically non-specific responses (i.e.in unsensitised individuals) in the bronchial tree. Micro-cystin-LR appears to be able to efficiently gain access tothe circulation by both intranasal and intratracheal routes[246-248], but Gram-negative bacterial endotoxin deliv-ered by inhalation does not cross into the pulmonary vas-culature to enter the circulation, and at least one



endotoxin-stimulated cytokine – TNF-α – is compartmen-talised in the airways [241,249,250]. What is open toquestion is whether the serious cases of pneumoniareported after recreational exposure to cyanobacteria (seeStewart et al [251]) may be explained by the induction ofan inflammatory response by inhaled cyanobacterial exo-toxin, which progresses to recruitment and activation ofneutrophils and is confined to the pulmonary alveolarcompartment. The possibility is also open as to whetherless dramatic reports of respiratory illness may also beexplained by a similar, albeit self-limiting process, inhealthy, non-atopic individuals. Of course, this specula-tion does not exclude the likelihood of different, overlap-ping mechanisms of disease that may explain thesephenomena – protein allergens in some cyanobacteriamay provoke symptoms in atopic individuals, such symp-toms possibly being exacerbated by the presence of cyano-bacterial and/or epiphytic bacterial endotoxins.

Cyanobacterial exotoxins may have the capacity to gener-ate respiratory illness in non-atopic individuals, withendotoxins from cyanobacteria or commensal bacteriapossibly augmenting the symptoms. The potential forcyanobacterial and/or contaminant endotoxin alone toproduce symptoms by inhalation exposure remains open,given the observation that LPS can produce measurableairway function changes in animal models and in somehealthy individuals [236,252-254]. Yet it remains unclearwhether such experimentally-induced changes in the air-way function of healthy volunteers correlate with symp-toms of respiratory dysfunction.

ConclusionLipid A, the endotoxic moiety of LPS, was in previous dec-ades thought to remain constant across different Gram-negative bacteria [100]. This is now understood to beincorrect; many non-enteric bacteria are seen to vary intheir lipid A structures. Because the biological activity oflipid A is determined by its structure, the toxic potential ofnon-enteric bacteria can vary. Gram-negative organismsoccupying different ecological niches will not have thesame requirements for growth, and their outer mem-branes can be expected to vary in order to meet differentenvironmental conditions [31]. Endotoxic potential can-not be assumed to be lacking in the LPS of non-entericbacteria, however, as seen in the high LPS agonist activityof lipid A from the non-pathogenic purple non-sulfur bac-terium Rubrivivax gelatinosus, as discussed above. A similarexample is given by another group of non-pathogenic bac-teria, Rhizobium spp., the LPS from some of which arecomparable to that of enterobacterial LPS in lethal toxicityand cytokine-inducing activity [255,256]. Determiningthe lipid A structures of various nuisance cyanobacteriawould be an interesting exercise in itself, but regardless ofthe findings, proponents of the "cyanobacterial LPS is

toxic" cause need to define plausible exposure routes toallow LPS to signal host receptors and initiate a patho-genic cytokine cascade.

From the discussion in this review, we will put thehypothesis that oral consumption of non-toxic cyanobac-teria, i.e. absolutely or essentially free of any of the knowncyanobacterial exotoxins, will not result in either vomit-ing or diarrhoea. This hypothesis would be falsified byexperiments that show isolated cyanobacterial LPS ornon-toxic crude extracts can cause gastrointestinal signsand/or pathology in a suitable model. Our impression isthat reports of G-I symptoms in humans exposed tocyanobacterial products are indications of innate defencesbeing signalled by exotoxins that have breached the intes-tinal barrier. Once this occurs, and gut permeability is suf-ficiently disrupted, LPS may well synergise the pathologyof cyanotoxins, especially the hepatotoxins. From whatlittle is known to date about the toxic potential of cyano-bacterial LPS, i.e. that they are weakly toxic compared tothose of the Enterobacteriaceae, gut-derived LPS wouldseem to be the more likely candidate for augmenting thepathology of cyanotoxins. In vivo studies of oral exposureto cyanotoxins would be well served by use of a vomiting-capable model, i.e. non-rodent experiments.

There does not appear to be good evidence that cyanobac-terial LPS are likely to initiate cutaneous reactions inhealthy people exposed in recreational or occupationalsettings. Cutaneous reactions to cyanobacteria are dis-cussed in detail elsewhere [257-259].

Exposure to bio-aerosols containing cyanobacterial endo-toxins may be worthy of investigation, but we are not con-vinced that cyanobacteria-related acute respiratory illnessin non-atopic, non-allergic individuals is not equally ormore likely to be explained by inhalation of cyanobacte-rial exotoxins. If some of the exotoxins turn out to possessligands that stimulate innate immune responses (dis-cussed further in [156]), then the large pool of residentalveolar macrophages would be prime candidates forinvolvement in respiratory defences. The outbreaks ofbath-water fever in Scandinavia and Africa (see accompa-nying review by Stewart et al [251]) were, in our opinion,suspicious of involvement by cyanobacterial exotoxinbreakthrough into reticulated supplies. Similar outbreaksin future should be vigorously investigated for cyanotox-ins if there is a suggestion of significant cyanobacterialcontamination of source water.

In conclusion, LPS of the Enterobacteriaceae are potentimmunomodulatory and immunotoxic bacterial productsthat stimulate a wide variety of responses in mammals,not least of these being a desire to wax lyrical on the topic.Thus:



"Endotoxins possess an intrinsic fascination that is noth-ing less than fabulous. They seem to have been endowedby Nature with virtues and vices in the exact and glamor-ous proportions needed to render them irresistible to anyinvestigator who comes to know them" [260].

And:

"The dual role of LPS as effector and target makes it a fas-cinating molecule which...still hides many miracles. Itintrigues at the same time clinical, biological, chemical,and biophysical researchers..."[83].

Facetiousness aside, these workers are pointing out thatthere is much to learn about the LPS of the most widelystudied Gram-negative bacteria, these being the Entero-bacteriaceae. The understanding of cyanobacterial LPS isutterly miniscule by comparison, and we urge cautionbefore continuing to attribute such a disparate range ofsymptoms in humans to contact with these materialswithout the required research evidence. Weckesser, Drewsand Mayer wrote in 1979 that:

"...the picture obtained with the Enterobacteriaceae can-not be assigned to other Gram-negative bacteria withoutdetailed investigations. Considering the broad spectrumin morphological and physiological diversity of the manytaxonomic groups of both photosynthetic bacteria andcyanobacteria, there is a wide open field for studies on thecomposition of their cell wall." [27].

Ressom et al [261] stated that:

"Given the enormous heterogeneity in LPS from Gram-negative bacteria there is every reason to suspect that thesame will apply to cyanobacterial LPS and, due to theirtaxonomic distance apart, cyanobacterial LPS are likely tobe different from those found in Gram-negative bacteria."

We agree with these statements.

AbbreviationsCYP cytochrome P450

DNA deoxyribonucleic acid

G-I gastrointestinal

GSH glutathione

GST glutathione S-transferase

IgE immunoglobulin E

IL interleukin

i.p. intra-peritoneal

LAL assay Limulus amoebocyte lysate assay

LBP lipopolysaccharide-binding protein

LD lethal dose

LPS lipopolysaccharide/s

SEs staphylococcal enterotoxins

Th cell T-helper cell

TLR Toll-like receptor

TNF-α tumour necrosis factor-alpha

Competing interestsThe author(s) declare that they have no competing inter-ests.

Authors' contributionsIS conducted the review; PJS and GRS supervised the workand contributed to redrafting the paper. All authors readand endorsed the final manuscript.

AcknowledgementsThanks to Dr Wasa Wickramasinghe for helpful discussions. This work was supported by grants from the South East Queensland Water Corporation and the Cooperative Research Centre for Water Quality and Treatment.

The National Research Centre for Environmental Toxicology is co-funded by Queensland Health, The University of Queensland, Griffith University and Queensland University of Technology.

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